For various care and treatment of mammal patients, it is necessary to determine concentrations of certain species in biological fluids. For instance, diabetics must be apprised of their blood glucose concentrations to enable insulin dosage to be adjusted. To determine blood glucose concentrations, blood is presently drawn several times per day by the diabetic, usually via a finger prick. If the blood glucose concentrations in such individuals are not properly maintained, the individuals become susceptible to numerous physiological problems, such as blindness, circulatory disorders, coronary artery disease, and renal failure. For these reasons, a substantial improvement in the quality of life of persons suffering from various maladies, such as diabetes mellitus, could be attained if the concentrations of species in body fluids are non-invasively and/or continuously determined. For example, for diabetic patients having external or implantable insulin pumps, a feedback loop for these pumps could be controlled by continuously monitoring glucose concentrations, to enable an artificial pancreas to be developed.
Exemplary systems have been previously proposed to monitor glucose in blood, as is necessary, for example, to control diabetic patients. This prior art is represented, for example, by Kaiser, U.S. Pat. No. 4,169,676, Muller, U.S. Pat. No. 4,427,889, and Dahne et al, European Patent Publication No. 0 160 768, and Bauer et al, Analytica Chimica Acta 197 (1987) pp. 295-301.
In Kaiser, glucose in blood is determined by irradiating a sample of the blood with a carbon dioxide laser source emitting a coherent beam, at a single frequency, in the mid-infrared region. An infrared beam derived from the laser source is coupled to the sample by way of an attenuated total reflectance crystal for the purpose of contacting the blood sample. The apparatus uses double beam instrumentation to examine the difference in absorption at the single frequency in the presence and absence of a sample. The reliability of the Kaiser device is materially impaired in certain situations because of the reliance on a single frequency beam for reasons explained below. Also, we have found from calculations based on available information that Kaiser's statement anent optical energy penetrating the skin to the depth of the blood capillaries is unlikely due to water absorption of the mid-infrared beam.
Muller discloses a system for quantifying glucose in blood by irradiating a sample of the blood with energy in a single beam from a laser operating at two frequencies in the mid-infrared region. The infrared radiation is either transmitted directly to the sample or by way of an attenuated total reflectance crystal for in vitro sampling. One frequency that irradiates the sample is in the 10.53-10.65 micrometer range, while the other irradiating frequency is in the 9.13-9.17 micrometer range. The radiation at the first frequency establishes a baseline absorption by the sample, while glucose absorption by the sample is determined from the intensity reduction caused by the sample at the second wavelength. The absorption ratio by the sample at the first and second frequencies quantifies the glucose of the sample. There is no glucose absorption at the first wavelength.
Dahne et al employs near-infrared spectroscopy for non-invasively transmitting optical energy in the nearinfrared spectrum through a finger or earlobe of a subject. Also discussed is the use of near-infrared energy diffusely reflected from deep within the tissue. Responses are derived at two different wavelengths to quantify glucose in the subject. One of the wavelengths is used to determine background absorption, while the other wavelength is used to determine glucose absorption. The ratio of the derived intensity at the two different wavelengths determines the quantity of glucose in the analyte biological fluid sample.
Bauer et al discloses monitoring glucose through the use of Fourier-transform infrared spectrometry wherein several absorbance versus wavelength curves are illustrated. A glucose concentration versus absorbance calibration curve, discussed in the last paragraph on p. 298, is constructed from several samples having known concentrations, in response to the intensity of the infrared energy absorbed by the samples at one wavelength, indicated as preferably 1035 cm.sup.-1.
All of the foregoing prior art techniques thus use only a single frequency analysis or ratio of two frequencies to determine a single proportionality constant describing a relationship between absorption of the infrared energy by the sample and concentration of a constituent of the biological fluid sample being analyzed, usually glucose. Hence, the prior art analysis is univariate since absorption by the constituent of interest at a single wavelength is determined.
However, univariate analysis has a tendency to be inaccurate in situations wherein there are concentration variations of any substance which absorbs at the analysis frequency. Biological systems are subject to numerous physiological perturbations over time and from person to person. The perturbations cause inaccuracies in univariate analysis, thereby decreasing the accuracy and precision of such analysis. The physiological perturbations involving any substance which absorbs at the analysis frequencies do not permit an operator of a system utilizing univariate analysis to recognize the resulting inaccuracy. In addition, nonlinearities may arise from spectroscopic instrumentation, refractive index dispersion, or interactions between molecules of the sample which cannot generally be modelled by univariate techniques. In addition, unknown biological materials in the sample have a tendency to interfere with the analysis process, particularly when these materials are present in varying amounts. Also the univariate techniques are usually not capable of identifying outlier samples, i.e., samples with data or constituents or spectra among the calibration or unknown data which differ from the remainder of the calibration set.
The described prior art systems utilizing midinfrared energy are not feasible for non-invasive in vivo determinations of glucose concentrations because of penetration depth limitations.
The most frequently employed prior art techniques for determining the concentration of molecular substances in biological fluids have used enzymatic, chemical and/or immunological methods. However, all of these techniques require invasive methods to draw a blood sample from a subject; typically, blood must be drawn several times a day by a finger prick, such as presently employed by a diabetic. For example, in the determination of glucose by diabetics, such invasive techniques must be performed using present technology. It would be highly desirable to provide a lessinvasive, continuous or semi-continuous system for automatically analyzing glucose concentrations in the control of diabetes mellitus.
It is, accordingly, an object of the present invention to provide a new and improved method of and apparatus for determining characteristics of a biological analyte sample.
Another object of the present invention is to provide a new and improved apparatus for and method of using infrared energy for analyzing biological fluids wherein the apparatus and method are particularly suitable for analyzing samples having concentrations of substances which variably or differentially absorb the infrared energy.
Another object of the invention is to provide a new and improved method of and apparatus for utilizing infrared energy to determine a characteristic, e.g., concentration, of a biological analyte by comparison of the absorption characteristics of said sample with a mathematical model constructed from several spectra of biological fluids having known absorption versus wavelength characteristics at known analyte concentrations.
A further object of the invention is to provide a new and improved apparatus for and method of analyzing biological fluids with infrared energy wherein interference with the infrared energy due to numerous physiological perturbations over time and between people does not have a particularly adverse effect on the results.
An additional object of the invention is to provide a new and improved apparatus for and method of using infrared energy to analyze biological fluids, wherein non-linearities due to various causes, for example, spectroscopic instrumentation, refractive index dispersion, and/or inter-molecular interactions, do not have an adverse effect on the analysis results.
An additional object of the present invention is to provide a new and improved apparatus for and method of using infrared energy to determine the nature of a biological sample wherein the presence of unknown biological materials in the sample does not interfere with the analysis of the sample, as long as these unknown biological materials are present in a calibration set which is used to derive a mathematical model which represents the response of known fluids to the infrared energy.
A further object of the invention is to provide a new and improved apparatus for and method of using infrared energy to determine characteristics of biological fluids wherein outlier samples subsisting in a calibration set used to derive a mathematical model are identified and either eliminated or accommodated so as not to have an adverse effect on the determination.
Another object of the invention is to provide a method of and apparatus for identifying the presence of outliers. The quality of the calibration results and the reliability of the unknown sample analyses can be critically dependent on the detection of outlier samples. In the calibration set, an outlier is a sample that does not exhibit the characteristic relationship between composition and the absorbance spectrum of the other calibration samples. During prediction, an outlier is a sample that is not representative of samples in the calibration set. Outliers in the calibration samples can impair the precision and accuracy of the calibration and limit the quality of the analyses of all future samples. The results of the analyses of outlier unknown samples by multivariate calibration cannot be considered reliable, and samples containing outliers should be analyzed by other methods. Thus, efficient detection of outlier samples is crucial for the successful application and wide acceptance of multivariate spectral analyses. For example, outliers occur as a result of changes in instrumental response, incorrect analyte determination by the reference method, unique type of sample, unexpected components, unusual baseline, incorrectly labeled or documented sample, etc.
Still an additional object of the invention is to provide a new and improved biological fluid analysis apparatus and method which is particularly adaptable, in certain embodiments, to non-invasive determinations.